Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen of compromised patients. Especially susceptible are individuals inflicted with severe burn wounds, surgery, eye complications, and cystic fibrosis. P. aeruginosa pathogenesis is facilitated by its ability to grow in hospital environments and its intrinsic resistant to antibiotics. P. aeruginosa produces a number of virulence determinants, which are either cell-surface components or secreted. The goal of this proposal is to define the role of exoenzyme S (ExoS) in P. aeruginosa pathogenesis. Recent studies have shown ExoS to be a bifunctional type-III secreted cytotoxin. Expression of the amino terminus of ExoS in eukaryotic cells stimulates actin reorganization, which involves small-molecular-weight GTPases of the Rho subfamily. Expression of the carboxyl terminus of ExoS, the ADP- ribosyltransferase domain, inhibits the activation of Ras by its guanine nucleotide exchange factor. Rho and Ras are molecular switches, which control numerous cellular processes, including wound healing, tissue regeneration, phagocytosis, and T cell activation. This proposal will address the molecular basis for the inhibition of signal transduction by ExoS, by determining the roles that ADP-ribosylation and cytoskeleton rearrangement play in the pathogenesis of P. aeruginosa. The hypothesis is that ExoS allows P. aeruginosa to establish an initial site of infection, through the inhibition of Ras- and Rho- mediated signal transduction pathways, which are essential for cell proliferation. Understanding the molecular properties of ExoS should provide insight into the development of vaccines and therapeutics to prevent the clinical manifestations of P. aeruginosa infections. The specific aims of this proposal are to: define the biochemical and cellular properties of ExoS and ExoT, measure in vitro and in vivo ADP-ribosylation of eukaryotic proteins by ExoS, and determine the molecular basis for actin reorganization elicited by ExoS.